A simple high-performance liquid chromatography (HPLC) method for the measurement of pyridoxal-5-phosphate and 4-pyridoxic acid in human plasma.

Background: Low concentration of plasma pyridoxal-5-phosphate (PLP) is associated with hyperhomocysteinemia and inflammation. Most methods for the measurement of plasma PLP require large specimen volume and involve the use of toxic reagents.
Methods: We have developed a HPLC method for the measurement of PLP and 4-pyridoxic acid (4-PA) in plasma, which requires small specimen volume. The samples are prepared without adding any toxic reagents. Furthermore, we have examined whether intake of vitamin B6 affects the concentration of plasma PLP and 4-PA.
Results: The coefficient of variation of the method was 6% and the recovery of the added vitamin in plasma was about 100%. The concentrations of plasma PLP and 4-PA in 168 healthy subjects were 40.6 (8.4-165.0) nmol/L, median and (range) and 17.5 (3.7-114.79) nmol/L, median and (range) respectively. In the multiple regression analyses, the concentration of plasma PLP was associated with the concentration of plasma 4-PA (p<0.0001), BMI, (p=0.02) and sex, (p=0.0008). The concentration of plasma 4-PA was associated with plasma PLP (p<0.0001), serum folate (p=0.004), smoking (p=0.03) and vitamin B6 intake (p=0.01).
Conclusion: The present method is suitable for large clinical studies for the measurement of plasma PLP and 4-PA. Our findings demonstrate that plasma 4-PA, BMI and sex are the major determinants of plasma PLP in healthy individuals.
(Copyright © 2014 Elsevier B.V. All rights reserved.)